title: Improving the spectral analysis of Fluorescence Resonance Energy Transfer in live cells: application to interferon receptors and Janus kinases.
abstract: The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, The University of Medicine and Dentistry of New Jersey, 675 Hoes Lane West, Piscataway, NJ 08855, USA. Electronic address: christopher.d.krause@gmail.com.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Krause
authors.forename: Gina
authors.initials: G
authors.lastname: Digioia
authors.forename: Lara S
authors.initials: LS
authors.lastname: Izotova
authors.forename: Sidney
authors.initials: S
authors.lastname: Pestka
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D053612:Janus Kinases / Q000032:analysis*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D046912:Multiprotein Complexes; D017471:Receptors, Interferon / Q000032:analysis* / Q000378:metabolism; D013194:Staining and Labeling; D000096743:Interferon gamma Receptor; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma; D053612:Janus Kinases; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor; D000097573:Red Fluorescent Protein
keywords: CFS; ChFP; CiFP; EBFP; ECFP; EGFP; ESaFP; EYFP; Equilibrium; FL; FLAG epitope (DYKDDDD); FP; FRET; IFN; IFN-α; IFN-γ; Interferon; Janus kinase; OFP; ORF; PEI; Receptor; StFP; TFP; confocal fluorescence spectroscopy; enhanced Sapphire fluorescent protein; enhanced blue fluorescent protein; enhanced cyan fluorescent protein; enhanced green fluorescent protein; enhanced yellow fluorescent protein; fluorescence resonance energy transfer; fluorescent protein; interferon; interferon-α; interferon-γ; mCherry/cherry fluorescent protein; mCitrine/monomeric citrine fluorescent protein; mOrange/orange fluorescent protein; mStrawberry/strawberry fluorescent protein; mTeal/teal fluorescent protein; open reading frame; polyethyleneimine